Vine regeneration, as a post-research technique, is of primary importance in the improvement of vines. Vine generation has a double objective in the wine quality and environmental protection fields. Regarding the latter aspect in particular, it is evident why it is very important to be able to regenerate and propagate, for example, plants that are resistant to certain diseases, so as to reduce the use of plant-protection products, which are often harmful to the environment.
With the aim of improving vine cultivars, while preserving the organoleptic characteristics of the products resulting therefrom, various in vitro culture and modification techniques have been tested especially so as to define early selection screens, as for example for resistance to diseases (BRANCHARD M., Agronomie, 1984 4 (9), 905-911) or for tolerance to the toxicity of certain elements in the soil (Bouquet A. et al., Symposium "Amelioration de la Vigne et Culture in vitro", (Vine improvement and in vitro culture), Paris, 1985, 147-157).
In order to develop such a vine regeneration technique, it is however necessary to have at least the following elements:
a stabilized culture of proembryogenic cells that can be replicated or modified genetically; PA1 a culture medium and a technique allowing its replication and its maintenance; and PA1 a culture medium and a technique that promote the formation of embryos and their development into plantlets.
In the present description, "plantlet" will be used to designate a plant derived from a somatic embryo, by analogy with the actual plantlet normally derived from the germination of a zygotic embryo of the seed. Still more precisely, the term "plantlet" used in the present description designates the plant in the state where, after complete development of the embryo, well developed green cotyledons and root elongation are observed.
Various cultural processes had been developed for the replication of the proembryogenic cells and the development of the embryos, especially by the formation and the development of secondary embryos, as described in U.S. Pat. No. 4,532,733.
However, the techniques described are faced with many difficulties, both with respect to the formation of the embryos, which are generally abnormal, and the development into plantlets, the latter being as a result obtained with particularly low yields.
In particular, it has been observed that the formation of secondary embryos, which are often abnormal, in fact led to a reduction in the yield of developed, that is to say cotyledonary, embryos at the end of the culture. Thus, these difficulties did not allow the use of these techniques subsequent to applied research results to be envisaged for industrial production.
Moreover, as is easily evident, the higher the embryogenic power of the cells of the culture used, the higher the yields of developed embryos and of plantlets.
Stabilized culture is understood to mean a culture of cells, or of cellular aggregates, of which the quantity of DNA per cellular nucleus remains identical during the course of replications, and which produces by means of regeneration techniques a plant identical to the initial plant, it being possible for such a stability to extend beyond 4 years of culturing and regular subculturings.
The cells are mainly diploid with the exception of those undergoing mitosis which are, in this case, tetraploid.
Variant or derived culture is understood to mean the cells modified especially by mutations, chromosome rearrangements, genetic recombination or epigenetic phenomena.
Comparable proembryogenic properties is understood to mean a culture capable of developing at least 100 embryos per mg of cells.